Effects And Mechanisms Of β-Elemene On Colorectal Cancer

Colorectal cancer occurs in the gastrointestinal tract,it is a common malignancy.It has no obvious symptoms in the early stages,it was found in the middle and late stages,tumors have metastasized and patient’s daily life is affected,bowel habits change,diarrhea,local abdominal pain,and blood in the stool occur,alternated diarrhea and constipation symptoms,and at late-stage occurs weight loss,weakness,anemia,and other systemic symptoms.The incidence and mortality of colorectal cancer account for the top 3 in digestive tract cancer,second only to gastric cancer and esophageal cancer.The treatment of colorectal cancer is based on surgery,combined with chemotherapy,immunotherapy,Chinese medicine treatment and other comprehensive therapies,so as to improve the surgical resection rate,reduce the patient’s recurrence rate,and obtain long-term survival opportunities.Chemotherapy is the main method for the treatment of colon cancer except surgery,but the low selectivity of chemotherapy drugs,toxic side effects,patients prone to recurrence and other shortcomings,making the treatment of colorectal cancer halted.Forcing researchers to actively seek effective and safe anti-colorectal cancer drugs.Elemene is an effective anticancer component extracted from Curcuma longa(Curcuma)of the genus Curcuma,and is used as a national second class antineoplasticnew drug in the treatment of cancerous ascites,pleural effusion,cerebral edema and solid tumors.β-Elemene is the main component of elemene,which has the effects of inhibiting tumor growth,inducing tumor cell apoptosis,inhibiting tumor invasion and metastasis,inhibiting tumor angiogenesis and reversing multi-drug resistance of chemotherapy.β-Elemene has high lipophilicity and small molecular weight,is unique to traditional anti-tumor drugs,making it easy to distribute in a variety of organs,especially its easier to cross the blood-brain barrier,and to a variety of tumors such as liver cancer,gastric cancer,breast cancer and brain cancer have obvious inhibitory effect.β-Elemene can be used alone or in combination with certain cancers to reduce side effects of chemotherapy and radiotherapy,improve patient survival,improve patient quality of life,reduce the risk of relapse and have good anti-tumor therapeutic prospects.There is little research on the inhibitory effect of β-elemene on colorectal cancer,and the specific mechanism and characteristics have not been revealed yet.The purpose of this project is to study the therapeutic effect ofβ-elemene on colorectal cancer and to explore the related molecular mechanism.Objective:To clarify the therapeutic effect of β-elemene on colorectal cancer and its related molecular mechanism.Methods:1.In vivo: BALB/c nude mice,male,42-48 days,and body weight 20-22 g,were injected subcutaneously with HT-29 cells in the right lower extremity to construct a xenografted model of colon cancer in nude mice.Normal group(not transplanted group),model group,β-elemene low dose group(25 mg/kg),β-elemene high dose group(50 mg/kg)and positive control 5-FU group(25 mg/kg),6 in each group,intraperitoneal injection,continuous administration of 21 days,β-elemene group administered once daily from Monday to Friday,positive control 5-FU group once a day,the normal group and the model group was given the same volume of 0.9% Na Cl solution.The body weight and tumor size of the nude mice were weighed every 2-3days.After 21 days,the nude mice were sacrificed by cervical dislocation,and the tumor was isolated and weighed.The heart,liver,spleen,lung and kidney were removed and weighed,fix in 10% formaldehyde solution.Calculate the tumor volume and tumor inhibition rate of nude mice in each group,make the tissue paraffin sections of HT-29 xenografts in nude mice.The difference of transplanted tumor in each group was observed by HE staining.The apoptosis of transplanted tumor was detected by TUNEL staining.The expression of Ki-67,Cleaved Caspase-3 and LC3 B were detectedby Immunohistochemistry.2.In vitro: Human colorectal cancer cell lines DLD-1 and HT-29 were selected for in vitro experiments.Different concentrations of β-elemene were added into DLD-1 and HT-29 cell culture medium.The effect of β-elemene on the cell viability of human colorectal cancer cell lines was detected by MTS assay,the effect of β-elemene on cell proliferation was detected by colony formation assay,the cell cycle was detected by flow cytometry,the effect of β-elemene on cell nucleus was detected by Hoechst33342 staining,the effect of β-elemene on cell membrane phosphatidylserine valgus by Annexin V/PI double staining,the change of cell mitochondrial membrane potential was detected by JC-1 staining,the formation of acidic autophagy cells was detected by acridine orange staining,apoptotic bodies and autophagosomes were observed by transmission electron microscopy,the level of ROS was detected by DCFH-DA staining,the expression levels of Cleaved Caspase-3,Caspase-3,Cleaved Caspase-9,Caspase-9,Cleaved PARP,PARP,LC3 B,SQSTM1,p-AMPK,AMPK,p-mTOR and mTOR protein was detected by western blot.Result:1.Effects of β-elemene on colon cancer xenografted nude mice.1.1 Evaluation of in vivo toxicity of β-elemene in HT-29 xenografted nude mice.The body weight of nude mice in the normal group increased first,then decreased and then increased,other groups increased and then decreased,and there was no significant difference in body weight between drug groups and the model group,indicated that β-elemene has no effect on body weight;the viscera index of each group of nude mice was compared with the normal group,the liver index of nude mice in the model group and drug groups were reduced due to inoculation of transplanted tumors,and no effect in other organs,there was no significant difference among the organs in drug groups and the model group,indicated that β-elemene has no effect on the organs;showed that β-elemene is not toxic to xenografts in nude mice.1.2 Effect of β-elemene on proliferation of HT-29 transplanted tumor in nude mice.The tumor volume gradually became larger,and the tumor cells were intact and well arranged in the model group.Compared with the model group,β-elemene(25mg/kg,50 mg/kg)could inhibit the tumor volume of in nude mice,the tumor inhibition rate increased,but the inhibitory effect is not obvious,reduced the expression of Ki-67 in tumor,tumor cell mass volume was significantly reduced Small,residual tumor cells arranged in sparse,tumor cells partially necrosis,results consistent with the positive drug 5-FU,indicated that β-elemene can inhibit the growth of HT-29 transplanted tumor in nude mice.1.3 Effect of β-elemene on apoptosis and autophagy of HT-29 transplanted tumor in nude mice.Compared with the model group,β-elemene(25mg/kg,50mg/kg)significantly increased the number of TUNEL positive cells,increased the expression of cleaved caspase-3,promoted the cleavage of caspase-3,increased the expression of autophagy-related protein LC3 B,indicated that β-elemene can induce apoptosis and produce autophagy of HT-29 transplanted tumor in nude mice.2.Effect of β-elemene on human colorectal cancer cells DLD-1 and HT-29.2.1 Effect of β-elemene on the proliferation of DLD-1 and HT-29 cells.The control group cells are intact and in good condition,compared with the control group,with the increase of β-elemene concentration(50,100,200,300,400,500,600 μmol/L),the cell survival number decreased,the cell gap became larger,the cell shrinks and the shape is faint,β-elemene(50,100,200,300 μmol/L)decreases the number of cell clones,the cell colony number gradually decreases,S phase and G2/M phase cycle block,indicated that β-elemene significantly inhibited the proliferation of human colorectal cancer cells.2.2 Effect of β-elemene on apoptosis of DLD-1 and HT-29 cells.Compared with the control group,β-elemene(50,100,200,300 μmol/L)made the cell nuclei of some DLD-1 and HT-29 cells became fragmented and nuclear chromatin was condensed,promoted the cell membrane phosphatidylserine valgus of DLD-1 and HT-29 cells,decreased the mitochondrial membrane potential,β-elemene(50,100,200,300 μmol/L)increased the expression level of apoptosis-related proteins Cleaved Caspase-3,Cleaved Caspase-9 and Cleaved PARP,the expression of Caspase-3,Caspase-9 and PARP proteins had no effect,promoted the cleavage of Caspase-3,Caspase-9 and PARP proteins,β-elemene(200 μmol/L)made HT-29 cells nuclear lysis,appeared apoptotic bodies,these results indicated thatβ-elemene can induce human colorectal cancer cells apoptosis.2.3 Effect of β-elemene on autophagy of DLD-1 and HT-29 cells Compared with the control group,β-elemene(300 μmol/L)made HT-29 cells appeared the autophagic vesicle bilayer membrane structure with mitochondria and lysosome structure,and β-elemene(50,100,200,300 μmol/L)promoted the increase of acid vesicle organelles(autophagy lysosome),β-elemene(100,200,300 μmol/L)increased the expression levels of autophagy-related proteins LC3 B and SQSTM1 in DLD-1 and HT-29 cells,these results indicated that β-elemene induces the formation of autophagosomes in human colorectal cancer cells and increases the level of autophagy.2.4 Effect of β-elemene on ROS levels,p-AMPK,AMPK,p-mTOR and mTOR protein expression in DLD-1 and HT-29 cells.Compared with the control group,β-elemene(50,100,200,300 μmol/L)increased the level of ROS in DLD-1 and HT-29 cells,β-elemene(100,200,300μmol/L)increased the expression of p-AMPK protein,decreased the expression of p-mTOR protein,no effect on the expression of AMPK and mTOR protein,indicated that β-elemene can promote human colorectal cancer cells ROS levels and AMPK protein phosphorylation,inhibit of mTOR protein phosphorylation.Conclusion:β-Elemene affects the proliferation,apoptosis and autophagy to inhibit the occurrence and development of colon cancer xenografts in nude mice,has anti-colon cancer effect,significantly inhibits the proliferation of human colorectal cancer cell lines DLD-1 and HT-29,induces apoptosis and autophagy in colorectal cancer cells,the effect of β-elemene on colorectal cancer was related to the increase of ROS level,the activation of AMPK and the decrease of mTOR phosphorylation.